Single‐cell transcriptomic analysis of PB and BM NK cells from severe aplastic anaemia patients

compensatory-related exhaustion. CIML NK cells may be reliable predictors of SAA treatment outcome. Exploration of the role of CIML NK in the occurrence and development of SAA will further clarify the immune pathogenesis of SAA and provide ideas for new therapeutic methods. In conclusion

cell dysfunction by proteomic analysis. 2 To further explore the subgroups of NK cells that may be implicated in the pathogenesis of SAA and find the differences between bone marrow NK cells (BM-NK) and peripheral blood NK cells (PB-NK), we conducted single-cell RNA sequencing (scRNA-seq) on BM-NK and PB-NK of SAA patients and healthy volunteers (HC). In keeping with our preceding studies, 1 the percentage of BM-NK was higher than that PB-NK in both HC and SAA, and the percentage of PB-NK and BM-NK were both markedly lower in the SAA than in the HC ( Figure S1). A total of 9280 cells (6134-13100) from each sample were sequenced, averaging 1876 genes analyzed per cell. After Harmony integration and Scanpy filtering, 97424 cells were included for further analysis, and 20 clusters were obtained from the initial UMAP (Unifrom Manifold Approximation and Projection) and Louvain clustering ( Figure S2). We removed the non-NK cell clusters (Table S1) and finally obtained eight groups of NK cells ( Figure 1A). These eight groups of NK cells were detected in all samples and passed quality control ( Figure S3). We didn't detect NCAM (CD56) in the dataset and used CD7/KLRD1/KLRF1/NGK7/GNLY as specific NK cell markers. 3 As expected, all these eight clusters expressed high levels of NK cell-specific markers ( Figure 1B). Referring to the previous literature of NK cell scRNA-seq, 3-6 combined with the characteristic marker genes of each cluster, we named these eight NK clusters as transitional NK, adaptive NK, CD56 bright NK, mature NK, cytokine-induced memory-like (CIML) NK1(normal CIML NK), CIML NK2(active CIML NK), active NK and terminal  (Table 1, Figure 1C,D, Figure S4). The cell number and the proportion of each NK cell subsets are shown Figure 1E and Table S3. We used RNA rate analysis 7 to describe the differentiation trajectory of NK cells ( Figure 1F) and found that terminal NK and CIML NK had unique transcriptional characteristics. CIML NK2 maybe derive from CIML NK1, and SCENIC analysis 8 also showed that these two clusters had similar regulons ( Figure 1G). What's more, the results showed that CD56 bright NK was in the early stage of NK cell differentiation, and transitional NK was a mixture of the intermediate process of NK differentiation, which is the transitional stage of CD56 bright NK, adaptive NK and active NK to mature NK.
After identifying the subgroup of NK cells, we analyzed the BM-NK and PB-NK transcriptomic changes in HC, identified 417 differentially expressed genes (DEGs) (Figure 2A). Note that 88.97% (371/417) of these DEGs were identified in only one cell types, and most of these genes were the characteristic genes of each cluster. Except for terminal NK cells, the gene expression of NK subsets in BM (bone marrow) and PB (peripheral blood) was basically similar, indicating that the function of BM-NK  Figure 2B). Terminal NK has active DNA replication ( Figure 2C). Interestingly, PB terminal NK cells had a stronger cytotoxic function and cell killing/ NK cell mediated cytotoxicity pathways were up-regulated, while BM terminal NK cells had stronger immune regulatory function, and immune-related path-ways were up-regulated ( Figure 2D,E). The results indicated that terminal NK cells with different localization have different functional tendencies.
Next, we identified 245 DEGs in BM-NK and 126 DEGs in PB-NK between SAA and HC ( Figure 3A). Of these DEGs, 44.9% and 47.6% DEGs appeared in multiple subgroups, and 58.73% of DEGs in PB-NK cells were consistent with BM-NK ( Figure 3B), suggesting that the same NK subgroup in different position (PB or BM) and different NK subgroups in the same position had partial similar functional changes under pathological conditions, especially in CD56 bright NK, transitional NK, adaptive NK and mature NK ( Figure S5). We found that, in SAA patients, both BM and PB-NK played a stronger immunomodulatory role, while the cytotoxic function was down-regulated, which may be disease-related functional depletion. What's more, in SAA BM-NK, we detected more up-regulation of immunomodulatory genes and more down-regulation of killing related genes and found several transcription factors and their ligands were up-regulated ( Figure 3C,D). Therefore, we believe that BM-NK was more sensitive than PB-NK in the disease state.
Finally, through Augur 9 analysis, we found that CIML NK1 and CIML NK2 were the most significantly cell clusters, which were contributed to disease phenotype in both BM and PB (BM: AUC (Area Under Curve) .815 and .810; PB: AUC: .725 and .772) ( Figure 3E). Both in PB and BM, the percentage of CIML NK cells of SAA patients was higher than that in HC, but the ratio of active CIML NK/total CIML NK was decreased ( Figure 3F). At the same time, the characteristic genes of BM and PB CIML NK cells in SAA were down-regulated and the pathways were enriched in cytokine stimulation and immune response ( Figure 3G). Further analysis showed that among these three SAA patients, those with a higher proportion of CIML NK cells and a higher CIML NK1/CIML NK2 ratio had a better prognosis ( Figure 3H, Table S4). So, we speculate that the increase proportion of CIML is a protective response in SAA, while the decrease in proportion and function of active CIML NK is a compensatory-related exhaustion. CIML NK cells may be reliable predictors of SAA treatment outcome. Exploration of the role of CIML NK in the occurrence and development of SAA will further clarify the immune pathogenesis of SAA and provide ideas for new therapeutic methods.
In conclusion, scRNA-seq can more accurately reflect the function of NK subsets and enrich our understanding of NK cells. BM-NK and PB-NK cells have similar functions, and BM-NK cells are more sensitive in the disease state. Further investigation is warranted to examine whether CIML NK cells are associated with treatment response. Additional samples and further analysis are needed to validate the results of different NK subsets.

S U P P O R T I N G I N F O R M AT I O N
Additional supporting information can be found online in the Supporting Information section at the end of this article.